Fluorescent dyes or stains have been widely used in biological detections in which the high detectability of fluorescence is desirable. By binding to a particular target in a biological sample enables the researcher to determine the presence or quantity of the target. Nucleic acids, such as DNA and RNA, are involved in the transmission of genetic information from one generation to the next, and the routine functions of live organisms. Nucleic acids are thus of interest and the objects of study. Fluorescent dyes that specifically bind to nucleic acids and form highly fluorescent complexes are useful tools for such study. These dyes can be used for detection of DNA or RNA in a variety of format, including in solution, in electrophoretic gels and blots, in microarray, dead or fixed cells, and live cells.
Several dyes are commercially available for detection of nucleic acids. Unsymmetrical cyanine dyes were described long before much was known about DNA, by Brooker, et al., J. AM. CHEM. SOC. 64, 199 (1942). The commercial dye Thiazole Orange has good applications in the quantitative analysis of immature blood cells or reticulocytes. U.S. Pat. No. 4,883,867 to Lee, et al. (1989); Lee, et al., Thiazole Orange: A New Dye for Reticulocyte Analysis, CYTOMETRY 7, 508 (1986). But, the limitation of Thiazole Orange is low detection sensitivity on nucleic acids. Ethidium bromide is the most widely used nucleic acid stain, and is commercially available from a number of suppliers. However, ethidium bromide is mutagenic, and its use requires significant care from the user to avoid contact with staining solutions, and special handing and waste disposal procedures (M. J. Waring, J. Mol. Biol. I 13, 269 (1965); McCann et al., Proc. Natl. Acad. Sci. USA, 72, 5135 (1975); and Fukunaga et al., Mutation Res. 127, 31 (19840)). PicoGreen is a stain selective for double stranded DNA and commercially available from Invitrogen; OliGreen is a stain useful for the quantitation of single stranded DNA and commercially available from Invitrogen; RiboGreen is a stain that is useful for quantitation of RNA in solution, and commercially available from Invitrogen; SYBR Green I is a stain selective for DNA and used for DNA gel stains and qPCR quantitation, and commercially available from Invitrogen; SYBR Gold is a high sensitive stain for both DNA and RNA, and commercially available from Invitrogen. These dyes are described in U.S. Pat. Nos. 5,436,134, 5,658,751 and 5,863,753. However, these dyes have low water soluability and have to be predisolved in organic solvent such as DMSO or DMF; and also have limited stability in aqueous solution, and have to be used within 24 hours before losing sensitivity. Another asymmetric cyanine dye, SYBR Safe, is commercially available from Invitrogen as an alternative to SYBR Green I and Ethidium bromide due to its low mutagenicity. This dye is described in U.S. Pat. Nos. 7,727,716 and 7,977,057. However, this alternative dye is less water soluability and low sensitive than desired.
Development of fluorescent dyes with improved water soluability and stability or the making or the use thereof is desirable.